JCB: Article
Published September 14, 2015
Structures of the yeast dynamin-like GTPase Sey1p
provide insight into homotypic ER fusion
Liming Yan,1* Sha Sun,2* Wei Wang,1,4 Juanming Shi,2 Xiaoyu Hu,2 Shiyan Wang,4 Dan Su,5 Zihe Rao,1,3,6
Junjie Hu,2,3 and Zhiyong Lou1,6
1School
of Medicine, Tsinghua University, Beijing, China 100084
of Genetics and Cell Biology, College of Life Sciences, Nankai University and Tianjin Key Laboratory of Protein Sciences, Tianjin, China 300071
3National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, China 100101
4State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, Henan University, Kaifeng, China 475004
5Collaborative Innovation Center for Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan
University, Chengdu, China 6100416Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu,
China 610041
Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases
(GTP­ases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle
domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This
domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with
GMP-PNP or GDP/AlF4− but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis,
even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our
results reveal unique features of Sey1p.
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THE JOURNAL OF CELL BIOLOGY
2Department
Introduction
The ER network consists of a continuous membrane system of
tubules and sheets (Shibata et al., 2006). ER membranes are
interconnected by homotypic fusion to maintain a reticular pattern and proper functioning. In metazoans, fusion is mediated
by a class of dynamin-like GTPases called atlastin (ATL; Hu
et al., 2009; Orso et al., 2009). In other eukaryotic organisms
lacking ATL, a similar class of GTPases, including Sey1p in
Saccharomyces cerevisiae (Hu et al., 2009; Anwar et al., 2012)
and RHD3 in Arabidopsis thaliana (Zhang et al., 2013), has
been identified. Deletion of Sey1p or depletion of ATL proteins in cells results in unbranched ER (Hu et al., 2009), delayed ER fusion (Anwar et al., 2012), or even ER fragmentation
(Orso et al., 2009). When purified and reconstituted in vitro,
members of both classes have been shown to mediate membrane fusion (Orso et al., 2009; Bian et al., 2011; Anwar et
al., 2012; Zhang et al., 2013).
GTPase-based ER fusogens play critical physiological
roles. In Candida albicans, deletion of Sey1p results in decreased virulence (Yamada-Okabe and Yamada-Okabe, 2002). In
A. thaliana, lack of RHD3 leads to short and wavy root hairs and
cell expansion defects (Schiefelbein and Somerville, 1990; Chen
*L. Yan and S. Sun contributed equally to this paper.
Correspondence to Junjie Hu: [email protected]; or Zhiyong Lou: louzy@mail.
tsinghua.edu.cn
Abbreviations used in this paper: 3HB, three-helix bundle; ATL, atlastin; AUC,
analytical ultracentrifugation; BDLP, bacterial dynamin-like protein; NBD, nitrobenzoxadiazole; PEG, polyethylene glycol; TM, transmembrane.
et al., 2011; Stefano et al., 2012), and the deletion of another family member causes lethality (Zhang et al., 2013). In Drosophila
melanogaster and Danio rerio, depletion of ATL causes neuronal
defects (Lee et al., 2008, 2009). In humans, mutations in ATL1,
the dominant form of ATL in the central nervous system, are
tightly associated with hereditary spastic paraplegia, a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity and weakness of
the lower limbs (Zhao et al., 2001; Salinas et al., 2008).
ATL, Sey1p, and RHD3 are anchored in the ER membrane by two closely spaced transmembrane (TM) segments,
exposing both termini to the cytosol. The mechanism for homotypic ER fusion has been partially unveiled using ATLs (Hu et
al., 2011; Lin et al., 2012). It is very different from SNARE or
viral protein-mediated fusion. Recent structural and biochemical studies indicate that the GTPase domain of ATL forms a
dimer upon GTP binding, initiating contact between apposing
membranes (Bian et al., 2011; Byrnes and Sondermann, 2011).
The region following the GTPase domain is a three-helix bundle (3HB) and can dock to its own GTPase domain or that of
a neighboring molecule, undergoing major conformational
changes during the GTP cycle to drive fusion (Bian et al., 2011;
© 2015 Yan et al. This article is distributed under the terms of an Attribution–Noncommercial–
Share Alike–No Mirror Sites license for the first six months after the publication date (see
http://www.rupress.org/terms). After six months it is available under a Creative Commons
License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at
http://creativecommons.org/licenses/by-nc-sa/3.0/).
Supplemental Material can be found at:
/content/suppl/2015/09/10/jcb.201502078.DC1.html
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 210 No. 6 961–972
www.jcb.org/cgi/doi/10.1083/jcb.201502078
JCB
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Published September 14, 2015
Figure 1. Structures of the cytosolic domain
of Sey1p. (A) Scheme showing the domains of
Sey1p from C. albicans (caSey1p) and S. cerevisiae (scSey1p). Residue numbers for each
domain are indicated. The domains of caSey1p are colored, respectively. 3HB, three-helix bundle; TMs, transmembrane segments; CT,
cytosolic tail. (B) Structure of the GDP/AlF4−bound form of caSey1p. The protomers in the
dimer are shown in colored (as in A) and gray
cartoon representation. The linker between the
GTPase and stalk domain and the long loop
in the stalk domain are highlighted. GDP is
shown in pink stick representation, AlF4− is in
cyan sticks, and magnesium ion is shown as a
lime sphere. The 3HBs in the stalk domain are
numbered, and the secondary structure elements are labeled. (C) As in B, but for structure
of caSey1p crystallized with GDP. (D) A topology plot of the stalk domain, colored as in A.
Results
Crystal structures of the cytosolic
domain of Sey1p
To investigate the mechanism of Sey1p-mediated membrane fusion, we determined the structures of the cytosolic domains (cytSey1p) of Sey1p from C. albicans (caSey1p). caSey1p shares
43% sequence identity with Sey1p from S. cerevisiae (scSey1p;
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see Fig. S1 A for sequence alignment). Residues 1–692 (truncation before the first TM region; Fig. 1 A) were expressed in Escherichia coli, purified, and crystallized first as an orthorhombic
form in the presence of GDP plus AlF4− (GDP/AlF4−). A structure at 2.8-Å resolution was determined by single anomalous diffraction (Table 1), and an electron density map of the nucleotide
is shown in Fig. S2 A. Only one caSey1p molecule was present
in the asymmetric unit, but a crystallographic dimer occurred
along one of the twofold symmetry axes, similar to the hexagonal crystal form of human ATL1 (hsATL1, form 1; Fig. S1 B).
CytSey1p consists of an N-terminal GTPase domain and
a long, stalk-like, helical domain connected by a linker region
(Fig. 1 B). In the dimer, the GTPase domains interact with one
another in such a way that the nucleotide binding sites face each
other. The linker regions of the two Sey1p molecules cross one
another, allowing a close association of the tops of the stalk
domains. The stalk domain is composed of four 3HBs, with the
last helix of each bundle extending into the first helix of the
next bundle (see Fig. 1 D for topology). The first 3HB (3HB-1)
is positioned under the GTPase domain of the pairing protomer.
If Sey1p ended here, it would resemble the configuration of the
crossover dimer of hsATL1 (form 1; Fig. S1, B and C). The
second and third 3HBs (3HB-2 and -3) pivot around each other,
so that the last 3HB (3HB-4) remains on the same side as the
GTPase domain. 3HB-4 is poorly ordered in general; a partially disordered long loop (residue 615–660) is found between
its second and third helices.
Nucleotide-dependent dimerization has been observed
with many dynamin-like GTPases (GTPase dimer and Bian et
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Byrnes et al., 2013). Membrane tethering has also been shown
to require continuous GTP hydrolysis of ATL (Saini et al., 2014;
Liu et al., 2015). Fusion is facilitated by a membrane-binding
amphipathic helix in the C-terminal tail and the TM segments
(Liu et al., 2012; Faust et al., 2015).
Identical membrane topology and conserved signature
motifs in the GTPase domains of ATL and Sey1p suggest that
Sey1p may behave similarly to ATL during membrane fusion.
However, the predicted helical domain between the GTPase
domain and TM segments is much longer (∼300 amino acids
more) in Sey1p compared with ATL. Furthermore, Sey1p is
concentrated in punctae along ER tubules, sometimes at threeway junctions of tubules, whereas ATL1 is mostly distributed
evenly on the ER membranes (Hu et al., 2009). Therefore, it is
unclear whether Sey1p acts the same way as ATL.
Here, we elucidate how Sey1p mediates homotypic membrane fusion. The proposed mechanism demonstrates a common
scheme for fusion mediated by dynamin-like GTPases and reveals
that the stalk domain of Sey1p possesses unique functional features.
Published September 14, 2015
al., 2011), but the configuration of the Sey1p stalk domain has
not been previously identified. The scale of the domain is comparable to that of bacterial dynamin-like protein (BDLP) and
is longer than Dynamin-1 or human guanylate-binding protein
1 (GBP1; Fig. S1 C). In BDLP structures, the stalk domain is
composed of two four-helix bundles connected by a flexible
hinge (Low and Löwe, 2006; Low et al., 2009). Major conformational changes, namely bending around the hinge, have been
proposed for BDLP. In Dynamin-1 structures, the stalk consists
of the bundle signaling element and one four-helix bundle, and
it plays critical role in oligomerization (Faelber et al., 2011;
Ford et al., 2011). The stalk of GBP1 starts with two 3HBs arranged similarly as 3HB-1 and -2 in Sey1p, followed by one
long helix that reaches back to the bottom of the GTPase domain (Ghosh et al., 2006). Unlike Sey1p, the stalk domains of
BDLP, GBP1, and Dynamin-1 interact extensively with their
GTPase domains, suggesting that the GTPase–stalk linkage
may have different roles in these proteins.
The same crystal form was obtained when GMP-PNP, a
nonhydrolyzable analogue of GTP, was added during purification
and crystallization. Overall, the Sey1p structure in GMP-PNP is
similar to that of GDP/AlF4−, with a root mean square deviation
of only 0.3709 Å (Fig. S1 C). In contrast, a different conformation of cytSey1p was observed when the protein was crystallized
with GDP (Fig. 1 C). This structure was determined from mono-
clinic crystals at 2.3-Å resolution using molecular replacement
with the GDP/AlF4− structure as a search model (Table 1). One
Sey1p molecule is found in each asymmetric unit, but unlike the
first structure, the stalk domain (mainly 3HB-1) is associated
with the GTPase domain of the same molecule. In one of the
crystallographic dimers, the GTPase domain of another Sey1p
molecule comes in close proximity (Fig. S1 E). Such a crystallographic “dimer” would be reminiscent of form 2 of hsATL1 (Fig.
S1 B), in which the 3HBs point in opposite directions and the
GTPase domains form a dimer interface of 670 Å2. However, this
Sey1p “dimer” buries only 455 Å2 of total surface area, suggesting less substantial contact between the two GTPase domains of
the symmetry mates. These results imply that, despite the overall structural similarity between Sey1p and ATL, these proteins
likely undergo nucleotide-dependent dimerization differently.
Nucleotide binding and GTP hydrolysis
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The GTPase domain of caSey1p folds in a similar manner as
other dynamin-like proteins. It is composed of a central β sheet
surrounded by six α helices. The nucleotide at the active site is
coordinated mainly by a magnesium ion and four signature motifs: the P-loop (G1, β1–α1), the switch 1 region (G2, α1–β2),
the switch 2 region (G3, β3–α2), and the G4 motif (after β5;
Fig. S2 B). Most residues involving GTP binding are conserved
between Sey1p and ATLs (Fig. S2 B). The two residues of ca-
Table 1. Data collection and refinement statistics
Parameters (data collection statistics)
GDP/AlF4−
GDP
GMP-PNP
SeMet
Native
a (Å)
75.2
76.27
228.1
76.6
b (Å)
120.6
120.5
43.6
120.9
Cell parameters
c (Å)
α, β, γ (°)
190.1
190.2
97.6
190.5
90.0, 90.0, 90.0
90.0, 90.0, 90.0
90.0, 108.1, 90.0
90.0, 90.0, 90.0
Space group
I212121
I212121
C2
I212121
Wavelength used (Å)
0.9792
1.0000
1.0000
1.0000
50.00 (3.05) – 3.00
50.00 (2.85) – 2.80
50.00 (2.42) – 2.30
50.00 (2.86) − 2.80
120307 (5,755)
224,007 (6,499)
122,150 (18,308)
139,072 (5,654)
17,527 (872)
21,774 (942)
40,802 (5,930)
19,897 (912)
Completeness (%)
98.7(99.0)
99.0 (87.5)
99.1 (99.4)
99.5 (94.7)
Average I/σ (I)
58.3 (24.4)
43.9 (4.4)
10.2 (2.3)
27.2 (2.9)
5.7 (10.8)
8.9 (45.2)
5.3 (44.3)
9.3 (58.6)
Resolution (Å)
No. of all reflections
No. of unique reflections
Rmerge (%)a
Refinement statistics
No. of reflections used (σ(F) > 0)
20,651
38,752
18,876
Rwork (%)b
25.3
22.0
24.4
Rfree (%)b
30.0
29.8
30.3
High resolution shell of Rfree (%)b
45.0
36.9
40.0
High resolution shell of Rwork (%)b
35.0
33.3
33.2
r.m.s.d. bond distance (Å)
0.018
0.015
0.013
r.m.s.d. bond angle (°)
1.695
2.000
1.852
Average B-value (Å2)
86.6
69.2
54.7
No. of protein atoms
5,446
5,327
5,348
No. of ligand atoms
34
58
66
No. of solvent atoms
126
146
22
Ramachandran plot
Res. in favored regions (%)
93.8
96.8
92.8
Res. in generously allowed regions (%)
5.5
3.0
5.9
Res. in disallowed regions (%)
0.7
1
1.2
Numbers in parentheses are corresponding values for the highest resolution shell. r.m.s.d., root-mean-square deviation; Res., residues.
aR
merge = ΣhΣl |Iih−<Ih>|/ΣhΣI <Ih>, where <Ih> is the mean of the observations Iih of reflection h.
bR
work = Σ(||Fp(obs)| − |Fp(calc) ||)/Σ |Fp(obs)|; Rfree is an R factor for a preselected subset (5%) of reflections that was not included in refinement.
Mechanism for ER fusion by Sey1p • Yan et al.
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Published September 14, 2015
Dimerization-induced membrane fusion
Nucleotide-dependent dimer formation is a common feature of
dynamin-like GTPases. ATL dimerization is physiologically
significant because ATL1 dimer mutants are defective in GTP
hydrolysis and membrane fusion, and one “hot spot” of the
hsATL1 mutations causing hereditary spastic paraplegia is localized in the GTPase domain near the dimer interface (Bian et
al., 2011). Sey1p dimerization has been reported (Anwar et al.,
2012) and was confirmed by our structural analysis (Fig. 1 B).
Notably, hsATL1 dimers were seen in all structures, regardless of which nucleotide was added (GMP-PNP, GDP/AlF4−,
and GDP), whereas caSey1p dimers only formed in the presence of GMP-PNP or GDP/AlF4−.
The dimerization of caSey1p involves a hydrophobic
patch on top of the GTPase domain, including a conserved
L257 (L233 in scSey1p and L274 in hsATL1) within the guanine cap, and several hydrophilic interactions along the GTPase interface (Fig. 2 A). The stalk domains also pack against
each other in the dimer. The closest association was in 3HB-2:
N407 and N410 from one molecule interact with K467 from
the other, and E417 makes a salt bridge with K475 (Fig. 2 B).
The bottoms of the stalk domains were also in proximity to
each other. However, no substantial interactions were seen. The
contact between the stalk domains occupies a surface area of
636 Å2, despite the length of the domain. The overall dimer
interface of caSey1p is 2891 Å2, with a total surface area of
36269 Å2 for each caSey1p molecule. In contrast, hsATL1
dimer buries 2,179 Å2 in form 2 or 3,677 Å2 in form 3 (with
949 Å2 contributed by the 3HBs), even when the total surface
area of each hsATL1 is ∼20,000 Å2 and the 3HB being much
shorter than the stalk domain of Sey1p.
To compare nucleotide-dependent dimerization of Sey1p
and ATL, we performed analytical ultracentrifugation (AUC).
As expected, purified cytSey1p from S. cerevisiae behaved as
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JCB • Volume 210 • Number 6 • 2015
a monomer in the absence of nucleotide or in the presence of
GDP, but it formed dimers in the presence of GMP-PNP or GDP/
AlF4− (Fig. 2 C, left). Sedimentation equilibrium experiments
revealed that the association of protomers was 57-fold stronger
with GMP-PNP than with GDP/AlF4− (Fig. S3 A). In contrast,
at the same protein concentration, cyt-hsATL1 exhibited much
more stable dimer formation when GDP/AlF4− was present compared with GMP-PNP (Fig. 2 C, right; and Fig. S3 B). The same
conclusion was drawn with the FRET-based assay (Liu et al.,
2015). These results imply that, unlike ATL, Sey1p relies more
on GTP binding than hydrolysis to achieve stable dimerization.
To probe the nucleotide requirement for Sey1p-mediated
fusion, we used an in vitro lipid-mixing assay. In this assay, purified full-length scSey1p was reconstituted into donor and acceptor vesicles as judged by flotation experiments (Fig. S4 A).
The donor vesicles contained lipids labeled with nitrobenzoxadiazole (NBD) and rhodamine at quenching concentrations; fusion with the unlabeled acceptor vesicles leads to fluorophore
dilution and dequenching. Wild-type scSey1p resulted in efficient fusion in the presence of GTP and Mg2+. No fusion was seen
with GDP or in the absence of Mg2+ (Fig. 2 D). Interestingly, the
addition of GMP-PNP caused moderate but reproducible fusion
(Fig. 2 D). Such activity was not observed for Drosophila ATL
(dmATL) in the presence of GMP-PNP or GDP/AlF4− (Fig. S4
C). Collectively, these results suggest that the linkage of opposing membranes through GTP binding-induced dimerization
may be sufficient to promote Sey1p-mediated fusion.
To confirm that GTP binding without hydrolysis can cause
fusion, we performed a visual assay of membrane tethering and
fusion. Wild-type full-length scSey1p was reconstituted into vesicles containing phospholipids with either a fluorescent Oregon
green or Rhodamine dye attached to the head groups. Samples
of the reactions were diluted and visualized with a confocal microscope. The vesicles initially appeared as green or red punctae
(Fig. 2 F, left). As recently reported for ATL, large yellow punctae (i.e., colocalization of the two dyes) appeared after the addition of GTP, indicating that the differently colored vesicles were
associated or fused with one another (Fig. 2 F, middle). When
EDTA, which chelates magnesium ions needed for nucleotide
binding, was added, some of these large punctae disappeared
or became smaller, indicating that some of the vesicles were
untethered (Fig. 2 F, right). Very little tethering was seen in the
presence of GDP/AlF4– (Fig. 2 H). In the presence of GMP-PNP,
a low level of fusion was observed and was not strongly affected
by EDTA addition (Fig. 2 G). These results confirmed that the
addition of GMP-PNP promotes some Sey1p-mediated fusion.
Next, we tested the effect of dimer mutations on Sey1p
activity. Mutation of L233 (equivalent to L257 in caSey1p
shown in Fig. 2 A) drastically reduced the dimerization (Fig. S3
C), GTPase activity (Fig. S2 F), and fusion activity of scSey1p
(Fig. 2 E and Fig. S4 B for reconstitution controls), whereas
mutation of the corresponding L274 in ATL1 resulted in weaker
defects in dimerization (Fig. S3 D). Deletion of 3HB-2 to
3HB-4 (residues 378–675) in the stalk domain (Δ3/4 stalk) of
scSey1p, which abolishes most of the stalk interactions in the
dimer and mimics the size of ATL1 (Fig. S1 C), strongly inhibited fusion activity (Fig. 2 E). scSey1p was also inactive when
3HB-2 was retained but 3HB-3 and -4 (residues 462–675) were
truncated (Δ1/2 stalk; Fig. 2 E). Conversely, the removal of a
long loop (residues 613–636) within 3HB-4 (ΔLoop) did not
significantly affect fusion activity (Fig. 2 E). Nevertheless, all
three mutations in the stalk domain had GTPase activity at wild-
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Sey1p’s G4 motif (R204 and D205) interact with the ribose of
the nucleotide (Fig. S2 C) and are only seen in a subclass of the
dynamin family that includes ATLs, Sey1p, RHD3, and GBPs.
However, S68 in the P-loop of caSey1p is equivalent to S41 in
Dynamin-1 (Chappie et al., 2010), which is required for GTP
hydrolysis (Fig. S2, B and D). When compared with dynamin-1,
the side chain of S68 does not point directly at the γ-phosphate
bond of GTP and does not rotate during the transition state.
Furthermore, a bridging sodium ion was found between S41 in
dynamin-1 and the nucleotide (Fig. S2 D) but was not seen in
Sey1p. In ATLs, an Arg finger (R77 in hsATL1) plays the same
role at the position of S68 in caSey1p (Bian et al., 2011; Byrnes
et al., 2013). This catalytic residue adopts a different rotamer
conformation after the cleavage of the γ-phosphate bond, which
in turn causes some minor local rearrangements (Fig. S2 D).
It is known that the GTPase activity of dynamin-1 is
stimulated more than 100-fold, when self-assembled on lipid
templates. Such dramatic increase was not seen with full-length
scSey1p, when its GTPase activity in detergent was compared
with that in reconstituted vesicles (Fig. S2 E). As expected and
similar to dynamin-1, mutant S47A in scSey1p (equivalent to
S68A in caSey1p) was less efficient in hydrolyzing GTP (Fig.
S2 F). In addition, the side chain of caSey1p T95 and the backbone nitrogen of T94 and T95 engage the nucleotide. Consistent with the structural observations, mutation of scSey1p T75A
(equivalent to T95A in caSey1p) reduces the enzymatic activity
of the GTPase (Fig. S2, C and F).
Published September 14, 2015
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Figure 2. Nucleotide-dependent dimerization of Sey1p. (A) The interface between the two GTPase domains. The cartoon representation is colored as in
Fig. 1. Key residues are indicated as sticks. The components of the second protomer (gray) are labeled with a prime symbol. (B) As in A, but for the interface
between the two stalk domains. (C) The size of cyt-scSey1p (40 µM, theoretical molecular mass 80.8 kD) or cyt-hsATL1 (40 µM, theoretical molecular mass
51.7 kD) was determined by AUC in the presence of the indicated nucleotides. The estimated molecular masses are given above the peaks in kilodaltons.
The data shown are from a single representative experiment out of three repeats. (D) Full-length wild-type (wt) scSey1p was reconstituted at equal concentrations into donor and acceptor vesicles (protein/lipid molar ratio of 1:200). Fusion of donor and acceptor vesicles was followed by the dequenching
Mechanism for ER fusion by Sey1p • Yan et al.
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Published September 14, 2015
type levels (Fig. S2 G). These results suggest that the length of
the stalk domain is important for fusion.
Conformational change induced by
Pi release
Analysis of Sey1p in mammalian cells
To analyze the function of Sey1p, we tested whether it can replace
ATLs in mammalian cells. We used siRNA to deplete the major
ATL proteins, mainly ATL2 and ATL3 in COS-7 cells, and visualized the ER morphology using calreticulin as a marker. Consistent
with previous results (Hu et al., 2009; Wu et al., 2015), when ATLs
were knocked down in COS-7 cells (Fig. 4 A), the ER appeared
as unbranched morphology in a majority of cells (Fig. 4, B and
D), indicating defects in ER fusion. As expected, wild-type human
ATL1, but not ATL1 with a mutation in the P-loop (K80A), efficiently restored the ER network in double-depleted cells (Fig. 4,
C and D; and Fig. S5 A). When scSey1p was expressed at a comparable level, most cells had normal ER morphology (Fig. 4, C
and D; and Fig. S5 A), suggesting that Sey1p and human ATL1
are equally active in maintaining the ER network. K50A (P-loop
mutant), L233A (dimer interface mutant), L297A, V298A (both
GTPase–stalk interface mutants), and Δ1/2stalk (stalk mutant) all
failed to rescue the defects in ER morphology (Fig. 4 D and see
Fig. S5 B for expression levels). Mutation M274A largely reduced
the percentage of abnormal ER (Fig. 4 D), likely because it only
affects the stabilization of the GDP-bound conformation and not
Sey1p dimerization. These results confirm that Sey1p plays an important role in in the maintenance of ER morphology.
To test whether caSey1p and scSey1p have similar functions
in cells, we expressed caSey1p in ATL-depleted COS-7 cells and
monitored ER morphology (Fig. 4 C). As expected and similar to
scSey1p, wild-type caSey1p but not GTP-binding defective mutant
K71A successfully replaced ATLs in mammalian cells (Fig. 4, C
and D). To further analyze the function of caSey1p, we expressed
caSey1p under the control of the endogenous SEY1 promoter in
a CEN vector (Fig. S5 C) in yeast cells lacking Sey1p and Yop1p,
an ER tubule-forming protein, and exhibiting abnormal ER morphology. caSey1p was mostly capable of substituting scSey1p in
the maintenance of ER morphology (Fig. S5 D). These results confirmed that caSey1p is a functional homologue of scSey1p.
Sey1p protein has been observed to be concentrated in
punctae along the ER tubules in yeast cells, mainly at the threeway junctions of the tubules (Hu et al., 2009). The localization of
scSey1p in COS-7 cells appears to be similar to that of yeast cells,
regardless of whether ATLs are present (Figs. 4 C and 5 A). Punctae were also seen when caSey1p was expressed in ATL-depleted
COS-7 cells (Fig. 4 C). Interestingly, scSey1p mutant with defective dimerization and fusion lost its punctate pattern (L233A;
of an NBD-labeled lipid present in the donor vesicles. Nucleotides, if indicated, were added at time 0. The data shown are from a single representative
experiment out of four repeats. (E) As in D, but with stalk mutants. Δ3/4 stalk, deletion of residues 378–675 in scSey1p; Δ1/2 stalk, deletion of 462–675;
ΔLoop, deletion of 613–636. The data shown are from a single representative experiment out of three repeats. (F) scSey1p-containing proteoliposomes,
which also contained either Rhodamine- or Oregon green–labeled lipids, were mixed at a 1:1 ratio (total lipid concentration: 0.6 mM). One aliquot was
analyzed immediately, a second was taken after incubation at 37°C with 1 mM GTP for 5 min, and a third was obtained after incubation with GTP followed
by addition of 10 mM EDTA. All samples were diluted, spotted onto a coverslip, and visualized by confocal microscopy. Bar, 5 µm. (G) As in F, but with
1 mM GMP-PNP. (H) As in F, but with GDP/AlF4−. Arrows indicate tethered/fused vesicles.
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The structures suggest that Sey1p retains its configuration
during hydrolysis of the γ-phosphate bond but undergoes at
least two conformational changes upon Pi release: one in the
active site and one involving repositioning of the stalk domain
relative to the GTPase domain. In the GDP/AlF4− or GMP-PNP
structure, switch 1 and 2 wrap around the nucleotide (Fig. 3 A).
On the other hand, both loops are distant from the active site
and partially disordered in the GDP structure (Fig. 3 A). These
movements of switch 1 and 2 are typical among GTPases and
explain why the dimer is stable with GMP-PNP or GDP/AlF4−
but not with GDP, as the switches are at the dimerization interface (Figs. 2 A and 3 A).
Absence of the γ-phosphate not only breaks the caSey1p
dimer but appears to alter the relative positions of the GTPase
and stalk domains (Fig. 3 B). When GMP-PNP or GDP/AlF4− is
bound, the linker connecting the GTPase domain and stalk domain attaches to the α7 of the other protomer. Major contacts
include L312 and L318 from one molecule, and V319 and F322
from the other molecule (Fig. 3 C). In addition, Q315 forms a homotypic interaction, and D309 from the linker makes salt bridges
to K323 and R376 of the other protomer (Fig. 3 C). In the GDP
state, α7 docks to α6 of the same Sey1p molecule using mostly
the same set of residues as in the GMP-PNP or GDP/AlF4− state.
However, V319 and F322 engage M295 in α6, Q315 hydrogen
bonds with Q303 and R299, and binding occurs between K323
and D291, and R376 and the carbonyl oxygen of A52 (Fig. 3 D).
A cross-linking assay was used to confirm the conformational change inferred from the structures. 3HB-1 of one cytSey1p molecule and α6 of the other molecule came in proximity
to one another in the presence of GMP-PNP or GDP/AlF4− but
not GDP or in the absence of nucleotide. As expected, a crosslinked dimer formed when the scSey1p mutant L297C/V298C
(L318C/V319C in caSey1p; see Fig. 3 C for positions of the
mutations) was treated with diamide to induce disulfide bridge
formation (Fig. 3 E, lanes 4 and 5 vs. 2 and 3). Cross-linked
products were not observed when the scSey1p mutant M274C/
V298C (M295C/V319C in caSey1p) was tested or when the reducing agent 2-mercaptoethanol was added (Fig. 3 E). Furthermore, as expected from the GDP/AlF4− and GMP-PNP structure
in which residue N407 of one caSey1p molecule comes in proximity to K467 of the other caSey1p molecule (Fig. 2 B), cysteines introduced at these positions formed a disulfide bridge
(Fig. 3 F, lanes 4 and 5). Finally, consistent with the AUC results, more dimers were formed with GMP-PNP than GDP/
AlF4− (Fig. 3, E and F, compare lanes 4 and 5).
To directly assess whether the conformational changes are
critical for fusion, we performed in vitro fusion assays. Mutation M274A in scSey1p (as M295A in caSey1p), which is
predicted to play no role in dimerization and localizes to the
GTPase-stalk interface in the GDP state, had reduced fusion
activity comparable to the level of wild-type protein with GMPPNP (Fig. 3 G). Similarly, mutant L297A in scSey1p (as L318A
in caSey1p), which appears to be important for dimerization but
not stabilization of the GDP state, was less effective in fusion
(Fig. 3 H). Finally, substitution of V298 (as V319 in caSey1p)
with ala, which disrupts GTPase–stalk interactions in both dimeric and monomeric conformations, drastically affected the
fusion activity (Fig. 3 H). Collectively, these data strongly support the conformations observed in the structures.
Published September 14, 2015
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Figure 3. Nucleotide-dependent conformational changes of Sey1p. (A) Cartoon representation of the active site. The GDP/AlF4− state is colored in red
and the GDP state in gold. The four signature motifs are highlighted. Note that switch 1 and switch 2 in the GDP state are partly disordered. The pairing
protomer in the GDP/AlF4− state is shown in gray. (B) Superposition of the Sey1p structures in the GDP/AlF4− state (red) and the GDP state (gold). The movement of the GTPase domain is highlighted. (C) The GTPase-linker interface in the GDP/AlF4− state. The cartoon representation is colored as in Fig. 1. Key
residues are shown in sticks. (D) As in C, but for the GTPase–stalk interface in the GDP state. (E) Purified cytosolic domains of scSey1p with cysteines at
positions 297 and 298 or at positions 274 and 298 were treated with the oxidant diamide in the presence of the indicated nucleotides. Non–cross-linked
protein (single asterisks) and cross-linked dimer (double asterisks) are indicated. Where indicated, the disulfide bridge was reduced with β-mercaptoethanol
(BME) before nonreducing SDS/PAGE. (F) As in E, but with wild type (wt) or mutant (cysteines at positions 407 and 467) of the cytosolic domain of caSey1p. (G) Fusion assays of the M274A mutant of scSey1p. Wild-type samples were included for comparison. (H) As in G, but with the mutants of L297A
and V298A. The data shown in G and H represents independent experiments with three independently purified batches of protein.
Mechanism for ER fusion by Sey1p • Yan et al.
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Fig. 5 B). ATL1 mainly distributed evenly along the ER tubules
(Fig. 5 C), but ATL3 formed punctae like Sey1p (Fig. 5 D). Given
that ATL3 was recently reported to be slower than ATL1 (Hu et
al., 2015), GTPases with relatively low hydrolysis activity should
tend to form punctae in cells. To test this possibility, we mutated
the Arg finger of ATL1 (R77) to Ala. It has been reported that
ATL1 R77A can still bind to GTP and form dimers, but GTP
hydrolysis occurs much more slowly (Byrnes et al., 2013). We
found that ATL1 R77A formed punctae in COS-7 cells (Fig. 5 E).
The same results were obtained when these proteins were expressed in ATL-depleted cells (Fig. S5 B). These data suggest
that punctae formation is dependent on GTPase activity.
Discussion
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Figure 4. Functional tests of Sey1p in cells. (A) COS-7 cells were transfected with siRNA oligonucleotides against hsATL2 and 3. The levels of
these proteins were determined by immunoblotting. GAPDH was used
as a loading control. IB, immunoblot. (B) The ER morphology of COS-7
cells was visualized using calreticulin, an endogenous luminal ER protein, and indirect immunofluorescence using a confocal microscope. (C)
Myc-hsATL1, Myc-scSey1p, or Myc-caSey1p was expressed in COS-7
cells and its localization determined by anti-Myc antibodies (green) and
compared with that of calreticulin (red) using indirect immunofluorescence
and confocal microscopy. (D) The ER morphology of indicated samples
was categorized as “normal” (as in the top image of B) or “unbranched”
(as in the bottom image of B). A total of 80–150 cells were counted for
each sample. All graphs are representative of at least three repetitions.
wt, wild type. Bars, 10 µm.
Our results suggest how Sey1p causes homotypic fusion of ER
membranes (Fig. 6 A). First, two Sey1p molecules in different
membranes bind GTP and form a dimer, thereby tethering the
membranes together. It is possible that the stalk domains in the
dimer do not associate initially but eventually reach the stable
side-by-side conformation. To efficiently juxtapose the two membranes, we speculate that the Sey1p dimer does not come in between the membrane but rather sits on the orbit of two closely
attached membrane regions with its GTPase domains pointing
away from the tethered area. Flattened, closely spaced membranes
were previously captured by cryo-EM during membrane tethering mediated by ATL or SNAREs (Diao et al., 2012; Hernandez et
al., 2012; Saini et al., 2014). Multiple dimers may self-associate,
perhaps through oligomerization of their TM regions and form
a ring to stabilize the tethered state, during which time the two
membranes begin to fuse. GTP hydrolysis and Pi release causes
a conformational change, probably triggered by changes in the
switch 1 region that is part of the GTPase domain dimer interface,
and the switch 2 region that propagates at the interaction surface
between the GTPase domain of one Sey1p molecule and the
linker and stalk domain of the other. The GTPase domain would
then be released and move freely until docking to its own stalk
domain, resulting in disruption of the Sey1p dimer. Regardless of
whether the membrane merging occurs before the monomerization of Sey1p, these molecules would start a new fusion attempt
by searching for a dimer partner on opposing membranes.
Surprisingly, we observed membrane fusion by Sey1p
even with GMP-PNP, indicating that fusion can occur without
GTP hydrolysis. However, most of the Sey1p dimers formed
with GMP-PNP would be on the same membrane, i.e., cis-dimer, and are futile. Fusion is much more efficient when GTP is
added, as GTP hydrolysis would allow Sey1p cycling between
dimer and monomer states. Consequently, more Sey1p monomers would be available for trans-interactions between different
membranes. Such competition between cis- and trans-interactions has also been recently reported for ATL (Liu et al., 2015).
Sey1p- and ATL-mediated fusion differs in that the power
stroke occurs at different steps of the GTP cycle. Stable trans-dimers of ATL (in form 3) are in a crossover configuration and
would not be possible without GTP hydrolysis (Fig. 6 B). To
achieve the tethered state, ATL molecules from apposing membranes have to bind GTP and dimerize into form 2 first; the two
GTPase domains can then rotate and recapture the 3HBs from
the pairing molecule to adopt form 3. The GTP-bound ATL
dimer may switch between the two conformations until GTP
hydrolysis drives the equilibrium toward form 3. Consistent
Published September 14, 2015
Figure 5. Sey1p punctae formation. (A)
Myc-scSey1p was expressed in COS-7 cells
and its localization determined by anti-Myc
antibodies and compared with that of calreticulin using indirect immunofluorescence and
confocal microscopy. The right image shows
an enlargement of the boxed region centered
on the tubular ER network. Bars, 10 µm. (B) As
in A, but with cells expressing Myc-scSey1p
L233A. (C) As in A, but with cells expressing
Myc-hsATL1. (D) As in A, but with cells expressing Myc-hsATL3. (E) As in A, but with cells expressing Myc-hsATL1 R77A. The arrowheads
indicate the puncta localized at the three-way
junctions of the ER network.
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Figure 6. Comparison of homotypic membrane fusion mediated by Sey1p and ATL. (A) A model of Sey1p-mediated fusion. See Discussion for details.
GTP and GDP molecules are indicated as cyan and blue spheres, respectively. (B) As in A, but for ATL. TMs, transmembrane segments; CT, cytosolic tail.
Mechanism for ER fusion by Sey1p • Yan et al.
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Materials and methods
Constructs, strains, and antibodies
The N-terminal cytosolic domains of C. albicans Sey1p (residues
1–692), codon-optimized S. cerevisiae Sey1p (residue 1–680), or
hsATL1 (residues 18–447) were inserted into the NheI–XhoI sites
of pET-28a (EMD Millipore), which includes a thrombin-cleavable,
N-terminal 6×His-tag. For fusion assay, codon-optimized full-length
scSey1p and full-length dmATL were cloned into the BamHI–XhoI
sites of pGEX6p-1 vector with a GST tag followed by a PreScission
protease digestion site at the N terminus. For mammalian expression,
Myc-scSey1p, Myc-caSey1p, or Myc-hsATL1 was PCR-amplified and
cloned into pcDNA4/TO (Invitrogen) by KpnI–BamHI, BamHI–Xhol,
or KpnI–Xhol sites. Myc-hsATL3 was PCR amplified and cloned into
pGW1. For endogenous level expression of scSey1p and caSey1p in
yeast cells, the coding region of scSey1p and caSey1p plus an N-terminal HA tag and 300 bp upstream and downstream sequences of
scSEY1 was amplified and inserted into the KpnI–NotI site of pRS316
(a URA/CEN plasmid). The plasmid pJK59, encoding Sec63-GFP was
used to visualize ER morphology. All point mutations were generated
using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies). The truncations (Δ3/4 stalk, deletion of residues 378–675
in scSey1p; Δ1/2 stalk, deletion of 462–675; ΔLoop, deletion of 613–
636) were generated using overlap PCR primers. All constructs were
confirmed by DNA sequencing.
The strain JHY4 (BY4741 sey1Δ::kanMX4 yop1Δ::HIS3MX6;
Hu et al., 2009) was used to visualize ER morphology. Mouse anti-Myc
and rabbit anti-Calreticulin antibodies were purchased from Abcam,
and mouse anti-HA and mouse anti–phosphoglycerate kinase antibodies were purchased from Sigma-Aldrich.
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Protein expression and purification
The cyt-caSey1p, cyt-scSey1p, and cyt-hsATL1 constructs were transformed into Escherichia coli strain BL21 (DE3), and cultures were
grown in Luria-Bertani media at 37°C to an OD600 of 0.6. Protein
expression was induced by the addition of 0.4 mM IPTG for 20 h at
16°C. Cells were harvested, resuspended in lysis buffer (20 mM Tris,
pH 8.0, 150 mM NaCl, 4 mM MgCl2, and 20 mM imidazole), and lysed
by an ultra-high pressure cell disrupter (JN-3000 PLUS; JNBIO). The
lysate was centrifuged at 16,000 rpm for 1 h, and the supernatant was
collected. The protein was isolated by Ni-NTA chromatography (GE
Healthcare) and further purified by ion-exchange chromatography (Resourse-Q or HiTrap Q; GE Healthcare) and gel filtration chromatography (Superdex-200; GE Healthcare). For crystallization, corresponding
nucleotides at 2 mM were included in the gel filtration buffer (Yan et
al., 2011; Chen et al., 2012).
For expression of selenomethionine-substituted (Se-Met) Sey1p,
cells were grown in M9 medium at 37°C to an OD600 of 0.6. Solid amino
acid supplements (50 mg/liter Lys, Thr, and Phe; 25 mg/liter Leu, Ile, and
Val; 30 mg/liter Se-Met) were added to the medium before induction. The
Se-Met protein was purified by the same procedure as the native protein.
Full-length dmATL or scSey1p were expressed and purified with
a GST tag. The proteins were solubilized and isolated in Triton X-100
(Anatrace), and the GST tag was cleaved before reconstitution (Bian et
al., 2011; Anwar et al., 2012).
X-ray crystallography
All native and selenomethionine-substituted proteins were concentrated to 8 mg/ml and crystallized by vapor diffusion methods at
16°C. Crystals of Sey1p with GDP/AlF4− were grown within days
in 160 mM lithium sulfate monohydrate, 80 mM Tris, pH 8.5, 20%
(wt/vol) polyethylene glycol (PEG) 3350, 40 mM ammonium acetate, 20 mM sodium citrate tribasic dehydrate, pH 5.6, 6% (wt/vol)
PEG 4000, and 20 mM spermidine. Crystals of Sey1p with GMPPNP appeared around 6 d in 120 mM dl-malic acid, pH 7.0, 16%
(wt/vol) PEG 3350, 20 mM bicine, pH 9.0, 400 mM magnesium
chloride hexahydrate, and 6% d-(+)-trehalose dihydrate. Crystals of Sey1p with GDP complex appeared around 2 mo in 20 mM
Tris, pH 8.5, 160 mM potassium sodium tartrate tetrahydrate, 4 mM
GDP, and 21% (wt/vol) PEG 3350.
All crystals were transferred to a cryo-protectant solution (4 M
sodium formate) before being flash frozen in liquid nitrogen for storage. The native data for Sey1p with GDP was collected in a beamline BL17U at the Shanghai Synchrotron Radiation Facility (China).
Other data were collected at 100 K using a charge-coupled device detector (Q270; Area Detector Systems Corporation) on beamline BL1A
at Photon Factory (Japan). All data were processed and scaled using
HKL2000 package 24 (Otwinowski and Minor, 1997).
The structure of Sey1p with GDP/AlF4− was solved by single anomalous diffraction of Se-Met using SHELX (Schneider
and Sheldrick, 2002). An initial model was built by the program
coot and used in molecular replacement to determine the structure
of Sey1p with GDP or GMP-PNP (Emsley and Cowtan, 2004).
For model refinement, the Phenix refinement program (phenix.refine) was used (Afonine et al., 2012). Interface and surface area
was calculated using PDBePISA.
AUC
Purified cyt-scSey1p and cyt-hsATL1 were used for AUC in a buffer
containing 50 mM Tris, pH 8.0, 300 mM NaCl, and 5 mM MgCl2.
Sedimentation velocity experiments were performed at 4°C in a proteomeLab XL-1 Protein Characterization System (Beckman Coulter).
2 mM GDP, 2 mM GMP-PNP, or 2 mM GDP/6 mM AlCl3/30 mM NaF
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with these models, ATL dimer is most stable when in complex
with GDP/AlF4−, whereas Sey1p dimer is strongest with GMPPNP. Such a difference would not be explained by the structures
but is likely determined by residues that sense the subtle change
in distance between β- and γ-phosphate.
The formation of punctae at the three-way junctions or
along ER tubules is seen in some, but not all, GTPase-based ER
fusogens. Our results suggest that the kinetics of GTP hydrolysis play a role in punctae formation. Slow GTPases, such as
Sey1p and ATL3, are prone to clustering. However, mutants with
further reduced activity, which do not sustain fusion, will lose
the punctate pattern and become evenly distributed on the ER
membrane. One scenario would be that effective fusion requires
the simultaneous participation of several GTPase molecules.
Alternatively, slow hydrolysis would prolong the existence of a
tethered complex, allowing the observation of punctae.
The proposed mechanism reveals that homotypic membrane fusion could be achieved by different approaches in
Sey1p and ATL but with a similar paradigm. Some of the mechanism could apply to RHD3 and its homologues in the fusion
of plant ER membranes, and to mitofusin/Fzo1p (Hermann et
al., 1998; Chen et al., 2003), another member of the dynamin
family, in the fusion of mitochondrial outer membranes. Three
point mutations were previously identified in RHD3 (Wang et
al., 1997). Based on sequence alignment with caSey1p, I134T
and D185N would reside near the GTPase dimer interface, and
A575V would be placed in the hydrophobic core of 3HB-4
(Fig. S1 D). The localization of these mutations confirms that
dimerization of the GTPase domain and integrity of the stalk
domain is critical for these dynamin-like proteins.
Published September 14, 2015
were added to 40 µM protein before centrifugation. All interference
data were collected at a speed of 42,000 rpm in a rotor (An-60 Ti; Beckman Coulter) and analyzed by the program SEDFIT in terms of a continuous c(s) distribution. In sedimentation equilibrium experiments, the
proteins were prepared at three different concentrations (0.2, 0.3, and
0.5 mg/ml). Interference data were collected at three different speeds
(8,000, 12,000, and 15,000 rpm for cytATL1; 6,400, 9,600, and12,000
rpm for cytSey1p) in a An-60 Ti rotor at 4°C and analyzed by SEDPHAT using a model of monomer–dimer self-association.
Cross-linking assays
1 µM wild-type or mutant cytSey1p protein in 50 mM Hepes, pH 7.4,
100 mM NaCl, and 5 mM MgCl2 with or without 1 mM GDP, 1 mM
GMP-PNP, or 1 mM GDP/3 mM AlCl3/15 mM NaF was incubated with
10 µM diamide at room temperature for 30 min 500 µM N-ethylmaleimide was used to quench the reactions. The samples were analyzed
by SDS-PAGE and Coomassie blue staining.
Fusion assays
Visual tethering/fusion assays
Full-length wild-type scSety1p was reconstituted into preformed
liposomes with Rhodamine-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) or Oregon green-DPPE (Invitrogen) in the
same way as described in the methods of Fusion assays. The vesicles
were mixed 1:1 in a reaction buffer containing 5 mM MgCl2. Different nucleotides were added and the samples diluted 1:100 in reaction
buffer or buffer with 10 mM EDTA. 5 µl samples were spotted onto a
glass coverslip and visualized by confocal microscopy. Oregon green
and Rhodamine dyes were excited with 488- and 561-nm lasers (100
mW), and their emissions selected with 530/40- and 620/50-nm filters,
respectively. All images were captured under room temperature on a
basic stand confocal microscope (AxioObsever Z1; Carl Zeiss) with a
63×/1.40 numerical aperture Plan Apochromat oil immersion objective
lens using ZEN2012 software (Carl Zeiss). Changes were made linearly across the entire image using Photoshop (Adobe).
GTPase activity assays
GTPase activity assays were performed with the phosphate assay
kit (Enzchek; Invitrogen). Reactions were performed in a 100 µl
volume with 1 µM wild-type or mutant protein as indicated by
the addition of 0.5 mM GTP. The absorbance at 360 nm was measured every minute over 30 min at 37°C using a micro-plate reader
(Synergy 4; BioTek). The rate of phosphate release was calculated based on a standard curve.
COS-7 cells were maintained at 37°C with 5% CO2 in DMEM containing
10% fetal bovine serum and passaged every 2–3 d. For siRNA transfections, COS-7 cells were plated at 50% confluency and transfected the next
morning with 13 nM of each siRNA oligonucleotide duplex using RNAiMAX (Invitrogen) for 6 h. Cells were then scraped for immunoblot analysis
or fixed for immunofluorescence analysis 48 h after transfection. Specific
siRNA oligonucleotides (Invitrogen) for ATL2 were targeted against the
following sequences: 5′-GGAGCUAUCCUUAUGAACAUUCAUA-3′;
and that for ATL3 were 5′-GGUUAGAGAUUGGAGUUUCCCUUAU-3′.
The control siRNA oligonucleotide was obtained from Invitrogen. The
siRNA-resistant constructs were transfected into knockdown cells using
Lipofectamine 3000 (Invitrogen) after 24 h of siRNA transfection.
Transfected cells were fixed for 20 min at room temperature with
4% formaldehyde (Bio Basic, Inc.) and permeabilized with 0.1% Triton
X-100 (Bio Basic, Inc.). The cells were washed twice with PBS and
probed with primary antibodies, including mouse anti-Myc antibody
(Abcam) diluted 1:500 or rabbit anti-calreticulin antibody (Abcam)
diluted 1:500, for 60 min in PBS containing 3% calf serum, followed
by incubation with various fluorophore-conjugated secondary antibodies for an additional 45 min (Alexa Fluor 488–conjugated anti–rabbit
or Alexa Fluor 594–conjugated anti–mouse (1:800 dilution; Invitrogen). For ER morphology rescue assay, yeast cells were imaged using
Sec63p-GFP as a marker (Wu et al., 2015). All images were captured
under room temperature on a confocal microscope (TCS SP5; Leica)
with a 63×/1.40 numerical aperture Plan Apochromat oil immersion
objective lens using LAS AF version 1.3.1 build 525 software (Leica).
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Lipid-mixing assays were performed as previously described (Bian et
al., 2011). In brief, lipids were dried down to a film, hydrated with
A100 buffer (25 mM Hepes, pH 7.4, 100 mM KCl, 1 mM 2-mercaptoethanol, and 10% glycerol), and extruded through polycarbonate filters with a pore size of 100 nm. Proteins were reconstituted at a protein/
lipid molar ratio of 1:200, except dmATL, which was reconstituted at
a ratio of 1:1,000 (Fig. S4 C). The flotation of proteoliposomes was
done on a sucrose gradient to determine the reconstitution efficiency.
20 mM proteoliposomes was mixed with 100 µl of 1.9 M sucrose and
overlaid with 100 µl of 1.25 M sucrose and 30 µl of 0.25 M sucrose (all
sucrose solutions were in 25 mM Hepes, pH 7.4, and 100 mM KCl).
After centrifugation at 174,000 g for 75 min and 4°C in a rotor (TLS55; Beckman Coulter), the gradient was fractionated into five fractions
and analyzed by SDS-PAGE and Coomassie blue staining. Donor and
acceptor liposomes were added at a 1:3 ratio. The initial NBD fluorescence was set to zero, and the maximum fluorescence was determined
after addition of dodecyl maltoside.
Mammalian cell culture, transfections, and microscopy
Accession numbers
The coordinates and structure factors have been deposited in the Protein Data Bank under accession nos. 5CA8 (GDP bound state), 5CA9
(GDP/AlF4– bound state), and 5CB2 (GMP-PNP bound state).
Online supplemental material
Fig. S1 shows comparison of Sey1p and other dynamin-like proteins.
Fig. S2 shows characterization of the GTPase domain of Sey1p. Fig.
S3 shows AUC analysis of Sey1p and ATL1. Fig. S4 shows controls for
fusion assay of Sey1p. Fig. S5 shows additional analysis of Sey1p in
cells. Online supplemental material is available at http://www.jcb.org/
cgi/content/full/jcb.201502078/DC1.
Ackowledgments
We thank T. Rapoport for critically reading the manuscript and the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for technical support.
L. Yan is a Tsinghua-Peking Center for Life Sciences postdoctoral fellow
and is supported by the China Postdoctoral Science Foundation
(2014M550713). Z. Lou is supported by the National Natural Science Foundation of China (grant 81322023) and the National Basic
Research Program of China (973 Program, grants 2013CB911103
and 2014CB542802). J. Hu is supported by the National Natural
Science Foundation of China (grant 31225006), the National Basic
Research Program of China (973 Program, grant 2012CB910302),
and an International Early Career Scientist grant from Howard Hughes
Medical Institute.
The authors declare no competing financial interests.
Submitted: 20 February 2015
Accepted: 5 August 2015
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